Streptococcus pneumoniae is a gram positive bacterium which is a major cause of invasive infections such as sepsis, meningitis, otitis media and lobar pneumonia (Tuomanen et al. NEJM 322:1280-1284, 1995). Infection by S. pneumoniae remains a significant health threat worldwide. Pneumococci bind avidly to cells of the upper and lower respiratory tract and to endothelial cells present in blood vessels. Like most bacteria, adherence of pneumococci to human cells is achieved by presentation of bacterial surface proteins that bind to eukaryotic cell surface proteins (Cundell, D. & Tuomanen, E. (1994) Microb Pathog 17:361-374). Pneumococci bind to non-inflamed epithelium, a process that can be viewed as asymptomatic carriage. It has been proposed that the conversion to invasive disease involves the local generation of inflammatory factors which, activating the human cell, change the number and type of receptors available on the human cells (Cundell, D. et al. (1995) Nature, 377:435-438). Presented with an opportunity in this new setting, pneumococci appear to take advantage and engage one of these up-regulated receptors. For example, bacteria translocate across cells of the respiratory tract via the polymeric immunoglobulin receptor (pIgR) (Zhang et al. (2000) Cell 102:827-837). Alternatively, when the bacteria are in the blood stream, the pneumococcal bacteria bind to endothelial cells, and the bacteria cross the blood vessel endothelium by binding to and transcytosing with the platelet activating factor (PAF) receptor (Cundell et al. (1995) Nature, 377:435-438). Within minutes of the appearance of the PAF receptor on activated cells, pneumococci undergo waves of enhanced adherence and invasion. Inhibition of bacterial binding to activated cells, for instance by soluble receptor analogs, or absence of the receptor blocks the progression to disease in animal models (Idanpaan-Heikkila, I. et al. (1997) J. Infect. Dis., 176:704-712; Radin et al. (2005) Infect. Immun. 73:7827-7835).
Pneumococci produce a family of proteins tethered to the bacterial surface by non-covalent association to the cell wall teichoic acid or lipoteichoic acid. This family of CBPs (choline binding proteins) is non-covalently bound to phosphorylcholine on the cell wall. CbpA, is a 75 kD surface-exposed choline binding protein that shows a chimeric architecture. There is a unique N-terminal domain, a proline rich region followed by a C-terminal domain comprised of 8 repeated regions responsible for binding to choline. CbpA binds specifically to pIgR on epithelial cells. When CbpA binds an extracellular domain present on pIgR, the pneumococcal bacteria are able to hijack the endocytosis machinery to translocate across nasopharyngeal epithelial cells into the blood stream. Mutants with defects in cbpA showed reduced virulence in the infant rat model for nasopharyngeal colonization and in mouse models of meningitis.
The choline binding domain was fully characterized by Lopez et al. in his studies of the autolytic enzyme (Ronda et al. (1987) Eur. J. Biochem, 164:621-624). Other proteins containing this domain include the autolysin of pneumococcus and the protective antigen, pneumococcal surface protein A (PspA) (Ronda, C. et al. (1987) Eur. J. Biochem., 164:621-624 and McDaniel, L. S., et al. (1992) Microb. Pathog., 13:261-269). CbpA shares the C-terminal choline binding domain with its other family members but its activity of binding to human cells arises from its unique N-terminal domain. Since the process of colonization and the progression to disease depend on pneumococcal attachment to human cells as a primary step, interruption of the function of the N terminal domain, either by cross reactive antibody or by competitive inhibition with a peptide mimicking this domain, may be critical to blocking disease.
The N-terminus of CbpA, corresponding to amino acid residues 39-514 of the CbpA protein from the Tigr4 strain, contains numerous repeats of the leucine zipper motif that cluster within 5 domains termed the A, B, R1, R2, and C domains. Domains A, B, and C are predicted to form coiled-coiled dimers. The R1 and R2 are also predicted to form self-associated, coiled-coil structures. The solution structure of CbpA was recently elucidated in Luo et al. (2005) EMBO J. 24(1):34-43, which is herein incorporated by reference in its entirety. In particular, the R2 domain of CbpA (amino acid residues approximately 327 to 442) was determined to comprise three anti-parallel alpha-helices. This three alpha-helix structure is similarly predicted for R1 domain, as was recently reported in Jordan et al. (2006) J. Am. Chem. Soc. 128(28):9119-9128.
Notably, the R domains from the Tigr4 strain of S. pneumoniae are highly conserved among CbpA sequences from other pneumococcal strains. Therefore, the R domains of CbpA are potentially important targets for the development of vaccines that are protective against numerous pneumococcal strains. Choline binding proteins for anti-pneumococcal vaccines are discussed in U.S. Pat. No. 6,858,706 and PCT International Application No. PCT/US97/07198, both of which are incorporated in their entirety by reference. Current vaccines against S. pneumoniae employ purified carbohydrates of the capsules of up to the 23 most common serotypes of this bacterium, but such vaccines are only 50% protective against pneumonia (Shapiro et al. NJEM 325:1453, 1991) and are not immunogenic under the age of 2. Conjugate vaccines are based on pneumococcal capsular carbohydrates linked to diphtheria toxoid or tetanus toxoid. Protection against pneumonia, sepsis, or meningitis for these vaccines is limited to the serotypes present in the formulation, thereby leaving patients unprotected against most of the ninety-two serotypes of this bacterium. Further, vaccines that are protective against both the colonization of pneumococcal bacteria in the nasopharynx as well as against entry of pneumococcal bacteria into the bloodstream are needed in the art. Therefore, the invention herein fills a long felt need by providing pharmaceutical compositions (e.g., vaccines) for the prevention and treatment of a wide range of serotypes of pneumococcal infections.